The objectives of this project are to develop molecular approaches to investigate pathogen-arthropod interactions of vector-borne diseases of human importance in the United States. The first objective was to develop a sensitive DNA hybridization probe to specifically identify Yersinia pestis, the causative agent of bubonic plague, in experimentally infected fleas. This was achieved in collaboration with K. McDonough and S. Falkow at Stanford University. Yersinia pestis infection in individual fleas of three species (Xenopsylla cheopis, Thrassis bacchi, and Diamanus montanus) were detectable using a 1000 base pair restriction fragment cloned from the pesticin plasmid. The same probe also detected Y. pestis in impression smears of spleen and liver of experimentally infected mice. A similar study has begun to develop a DNA probe to detect Rickettsia typhi, the causative agent of murine typhus, in fleas. Protocols for the purification of murine toxin and Fraction 1 antigen of Y. pestis are being developed to provide reagents for alternative methods for the rapid detection of Y. pestis in infected fleas.